Regulatory role of cAMP on expression of Cdk4 and p27(Kip1) by inhibiting phosphatidylinositol 3-kinase in corneal endothelial cells.

Abstract

Fibroblast growth factor (FGF)-2 is a potent mitogen of corneal endothelial cells (CECs). Results in an earlier study showed that FGF-2 activates phosphatidylinositol (PI) 3-kinase to stimulate the cell cycle machinery. The current study was designed to determine whether adenosine 3',5'-monophosphate (cAMP) antagonizes FGF-2 by inhibiting PI 3-kinase/Akt pathways, thus leading to regulation of cyclin-dependent kinase 4 (Cdk4) and p27(Kip1) (p27) expression. Cell proliferation was assayed by counting cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of Cdk4, p27, PI 3-kinase, Akt, and beta-actin was analyzed by immunoblot analysis. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. 8-Bromoadenosine cAMP (8-Br-cAMP), a diffusible cAMP analogue, inhibited the PI 3-kinase/Akt signaling pathways. The 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels. They also equally inhibited cell proliferation, nuclear translocation of Cdk4, and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP was mediated by a direct inhibition of PI 3-kinase activity, which subsequently blocked phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP promoted a rapid turnover of Akt protein, and 8-Br-cAMP markedly reduced the half-life of Cdk4 protein. This inhibitory activity of cAMP was not mediated by PKA, but 8-Br-cAMP inhibited membrane localization of the p85 regulatory subunit of PI 3-kinase. These data support the hypothesis that cAMP inhibits the proliferation of CECs, preventing them from entering the S phase by negatively regulating PI 3-kinase.